High-Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into the Catalytic Mechanism and Inhibition by Aldehydes,

Abstract

Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD+ in most prokaryotes, most single cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD+ homeostasis has increased interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD+ consuming enzymes, such as the NAD+-dependent deacetylases (sirtuins). Here, we report several high resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. The structure of the C136S mutant in complex with nicotinamide provides details about substrate binding while a trapped nicotinoyl-thioester complexed with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features including a metal ion that coordinates the substrate and the catalytically relevant water molecule, and an oxyanion hole which both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence for several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.

Structural and Kinetic Isotope Effect Studies of Nicotinamidase (Pnc1) from Saccharomyces cerevisiae

“…In addition to the covalent linkage to C167, nicotinaldehyde is ligated to the active-site zinc through the pyridine ring nitrogen (Figure 1). This zinc ligation is consistent with recent structures of nicotinamidases from Acinetobacter baumanii and Streptococcus pneumoniae (31, 52). The active-site zinc is also ligated by D51, H53, and H94, consistent with the previous S. cerevisiae Pnc1 apo-structure (32).…”

“…Furthermore, we observed evidence for two water molecules that also coordinate the zinc; one of these is within hydrogen bonding distance of one conformation of E129 (E129 is present in two conformations in the structure) (Figure 1C). The carbonyl oxygen of nicotinaldehyde forms hydrogen bonds to the backbone amide nitrogens of A163 and C167 consistent with recent structures of S. pneumoniae PncA (52). The pyridine ring of nicotinaldehyde is also bound within an aromatic cage consisting of F13, W91, Y131, and Y166 (Figure 1B).…”

“…These Pnc1 mutants were chosen because of the differences observed in the determined kinetic parameters and their ability to achieve adequate product conversion to determine KIEs. These KIEs permitted us to investigate whether the mutated residues are responsible for the actions discussed here and by others (30, 31, 33, 52, 55). For the Zn 2+ ligation mutant, D51N, the KIE values were both lower than for Pnc1 WT, whereas a higher 15 N KIE and a lower 13 C KIE was observed for D51A compared to Pnc1 WT.…”

Combined Whole-Cell High-Throughput Functional Screening for Identification of New Nicotinamidases/Pyrazinamidases in Metagenomic/Polygenomic Libraries

“…HL-48 (UniProt code: A0A0P7YXE9). In addition, PolyNic also showed variable similarity (25–46%, Supplementary Table S1) with the crystallized nicotinamidases found in the bibliography ( Figure 5 ) (Du et al, 2001; Hu et al, 2007; Fyfe et al, 2009; French et al, 2010a; Gazanion et al, 2011; Liu et al, 2011; Petrella et al, 2011). …”

A Key Enzyme of the NAD + Salvage Pathway in Thermus thermophilus: Characterization of Nicotinamidase and the Impact of Its Gene Deletion at High Temperatures

“…S2). The kinetic parameters of nicotinamidase have been studied in different bacteria (Mycobacterium tuberculosis [17], Streptococcus pneumoniae [18], and Oceanobacillus iheyensis [16]), archaea (Acidilobus saccharovorans [19]), and eukaryotes (Saccharomyces cerevisiae [12]). The kinetic parameters determined in this study are comparable to those of nicotinamidases previously reported from other organisms ( Table 1).…”